Project Summary/Abstract Respiratory Syncytial Virus (RSV) is a ubiquitous respiratory illness, and the leading viral cause of infant mortality worldwide. Despite its prevalence, there remains no effective vaccine, nor an efficacious RSV-specific therapeutic for infected patients. It is critical that we understand the fundamental biology underlying RSV infection and the subsequent host response to further our understanding of this pathogen, and lay the foundation for novel therapeutic strategies. The temporal dynamics of RSV infection have been characterized in bulk studies in vitro, in vivo, and ex vivo, however, these bulk studies likely underestimate the complexity and heterogeneity of the host respiratory epithelial response to infection. I have performed preliminary experiments to investigate the single cell response of respiratory epithelial cells (A549 cells) to RSV infection and my data revealed viral burden dependent and distinct gene networks activated over a twelve hour course of RSV infection. My data suggests a model in which infection may be considered a dynamic process with a spectrum of transcriptional states indicative of infection state. Indeed, I find expression of interferon stimulated genes (ISGs) is stronger in cells with undetectable RSV transcripts, compared to nearby infected cells. This is indicative of a compromised innate defense state within infected cells, but sensed by the surrounding cells. Given my preliminary studies, I propose a research strategy that seeks to test my hypotheses that the proinflammatory response during infection is largely driven by nearby uninfected cells, and that distinct proinflammatory gene signatures will correlate with different stages of the adjacent viral life cycle. In Aim 1, I will characterize the viral and host single cell temporal transcriptional dynamics of RSV infection in both infected and uninfected bystander cells, and produce single cell trajectories modeling RSV infection. Additionally, I will determine the contribution of paracrine signaling to the proinflammatory response initiated after RSV infection, by blocking protein secretion with Brefeldin A, and investigating the transcriptional response of uninfected bystander cells. In Aim 2, I will complement this study by performing an ISG-wide CRISPRa activation screen to identify host factors that influence both RSV replication and virion production. The completion of this study, which leverages single cell sequencing, bioinformatics, and CRISPR screening techniques, will provide pivotal insights into our understanding of the antiviral response to RSV infection.